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Study on Mechanical Property and Biocompatibilityin Vitro of 3D Printing Tantalum- Niobium AlloyImplants.Huiling Li 

Central South University Xiangya School of Medicine https://orcid.org/0000-0003-2777-990XJian Zhang 

Z-BEST Dental Science and Technology CO.LTD, Hunan.Zhigang Yao 

Central South UniversityJunhui Huang  ( huangjunhuixy@163.com )

Central South University Xiangya School of Medicine

Research

Keywords: 3D-printing, SLS, Tantalum-Niobium alloy, implant, human Oral Mucosa Fibroblast,mechanical property, biocompatibility

Posted Date: February 26th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-249911/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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Abstracttooth defects or deletions. Tantalum (Ta) has been widely used in the biomedical �eld, but its applicationin arti�cial dental implants is rarely reported. In this study, Tantalum-Niobium alloy (TaNb40) implantprepared by 3D printing—SLS was used as the research subject. Its mechanical properties in vitro and thebiocompatibility by utilizing human Oral Mucosa Fibroblasts (hOMF) were studied.

Results: The mechanical property test results were that the Tensile Strength, Yield Strength, Elongation,and Vickers hardness of TaNb40 implant was 548 ± 50MPa, 420 ± 30MPa, 40%, and 425HV, respectively,manifesting the two indicators met the requirements of dental metal implant, and had good mechanicalproperties of wear resistance and not easy to brittle fracture. The cytocompatibility test showed thatTaNb40 did not inhibit cell proliferation and produce cell cycle arrest. The cytotoxicity grade was 1, therewas almost no metal ion release in the culture medium, and the surface had �ne cell early adhesion,which met the requirements of biomedical materials.

Conclusion: The TaNb40 prepared by domestic 3D printing—SLS has excellent mechanical properties andcytocompatibility in vitro, which is expected to replace the Titanium metal implant prepared by thetraditional casting method, having a leading signi�cance for the formation of implants with independentintellectual property rights. 

BackgroundThe development of medicine and materials science largely depends on people's continuous pursuit oflife quality and life expectancy. The defects or deletions of certain tissues or organs are caused by somediseases, trauma, and other factors. To restore their morphology and physiological function, biomedicalmaterials are needed to repair the damaged tissues. Among them, bio-metallic materials are goodsubstitutes for the defects in the body, which have high mechanical strength, stable chemical properties,and excellent biocompatibility. At present, metal materials are mainly used in arti�cial joints, internal�xation of trauma, spine and orthopedic restoration, etc. Three widely used biomaterials in orthopedicsinclude Stainless Steel (SS), Cobalt-based alloy, and Titanium (Ti) alloy [1]. According to the number andtype of dentition defects or deletions, different prosthodontics can be adopted, including �xed denture,removable denture, and implant restoration. Implant restoration, a permanent structure, is an importantway to replace the missing teeth, the system mainly consists of two parts: implant and abutment. Theabutment is fastened to the implant by a screw, and provides a �xed point for tooth restoration [2].Therefore, it not only has the advantages of good retention, stability, high masticatory e�ciency, and nodamage to other teeth but also beautiful appearance and comfort, which has become a popular repairmethod for clinicians and patients. The abutments can be made of different materials, including Ti, Gold,Zirconia, Alumina, and polymer [3]. In the late 1960s, the �rst batch of ceramic dental implants made ofAlumina was developed, however, due to its poor mechanical properties, it was easy to fracture whenloaded with external force, and �nally was driven out of the market [4-5]. At present, most dental implantsare composed of commercially pure Ti, which is a highly biocompatible material with a survival rate of

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90.9% to 97.7% after 15 years of implantation [6-8]. The high success rate is partly owing to theformation of oxide �lm (mainly TiO2), which separates the base metal from the tissue and provides asuitable surface for osseointegration [9]. Although the excellent performance of Ti implants is attributedto surface properties to some extent, the mechanical properties may not be su�cient in applicationsrequiring narrow diameter, short implants, or exposure to excessive occlusal stress [10]. Therefore, for thesake of improving the osseointegration and long-term survival rate of implants, many researchers focuson the development of multifunctional Ti surfaces. For example, in addition to sandblasting and acid-etched surfaces, there are some scienti�c reports on the survival rates of the Branemark dental implantsystem (anodized TiO2 surface) and Astra Tech dental implant system (TiO2 surface / sandblasting),and the biological activity of Ti is improved by adding bioactive microelement [11-12]. In recent years,Titanium-Zirconium (TiZr) alloy and Zirconia (ZrO2) have become alternative materials due to their highmechanical strength and low corrosion sensitivity [13]. TiZr alloy is a promising candidate material,which can also reduce the cytotoxicity related to Ti alloy other components, especially Aluminum (Al) andVanadium (V) [14-15]. The natural oxide �lm of Zr increases its passivation, compared with pure Ti, therelease of metal ions is signi�cantly reduced when immersed in acid medium of the simulated oralenvironment [16]. Although TiZr alloy has improved its corrosion resistance, it is similar to Ti substrate interms of initial bacterial attachment and bio�lm formation [17]. Like Ti, ZrO2 also presents an oxidesurface, which is conducive to bone integration similar to Ti [18-19]. However, accelerated aging in thewater environment will lead to partially stable ZrO2 oxide �lm degradation[20-21]. Because the successrate of implant implantation is related to many factors, such as bone quality, implant interface properties,stress distribution around the implant, so the research and development of new implant materials need tomake up for the de�ciencies of the current materials in terms of biomechanical properties. Moreover, aswe all know, China has a large population, the dental implants and other high-quality materials mainlyrely on imports. Besides, there are some metal and alloy dental implant materials in the Chinese market,existing different degrees of histocompatibility and toxic side effects of alloy elements on the humanbody, as well as, the variety is single, and the price is expensive. One of the key goals of life science inChina's medium and long-term science and technology development plan is to develop new-typebiomedical materials such as individualized medical engineering technology and human tissue organreplacement.

Tantalum (Ta) is a transition group element, which belongs to the VB group in the periodic table. Itsmelting point is as high as 2995 ℃, Ta alloy has excellent thermal shock resistance and moldingtoughness. Besides, it also has high thermal and electrical conductivity. Therefore, Ta is widely used inelectronics, chemical industry, aviation, weapon system and other aspects [22-24]. In the air and othermedia, the Ta surface layer will react with oxygen or other oxidants to form an oxide �lm, which has amore compact structure and insulation performance, and excellent corrosion resistance [23]. As early asthe middle of the 19th century, the biomedical �eld began to use this metal to prepare pacemakerelectrode materials, nerve repair �lms, and head modeling versions, etc [25]. Stable biologicalcharacteristics make Ta play an important role in the medical �eld, such as Ta nail for the treatment ofearly adult femoral head necrosis, Ta metal rod supporting bone tissue defect, the trabecular metal used

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in joint and spinal surgery, etc. [26-29]. At present, Porous Tantalum Trabecular Metal (PTTM) has beenincorporated into Ti alloy dental implants to improve osseointegration in many studies, but Ta as anarti�cial root is rarely reported. Solid pure Ta has high mechanical strength and density (16.68g/cm3),therefore, porous structure involving porosity and pore diameter has obvious advantages in this aspect.By adjusting the porosity and pore size of the material, the "stress shielding" is reduced or eliminated, sothat facilitating its elastic modulus to match that of human bone tissue. However, high meltingtemperature, a strong a�nity for oxygen, di�culty in material preparation, and high cost make it verydi�cult to prepare Ta through ordinary methods, which limits the wide application of Ta [30]. Ta andNiobium (Nb) are geared to the same category in the periodic table of elements. Nb has a lower meltingtemperature (2477℃), elastic modulus, and raw material cost than Ta and Nb is a strong β stabilizer [30-31]. Therefore, the Ta-Nb alloy has a lower elastic modulus than the Ta scaffold, without inhibiting itscorrosion resistance or introducing any cytotoxic components, and reducing the manufacturing cost.

3D printing is a highly adjustable and complex device matching with human anatomy position, which issuitable for patients and has become a leading manufacturing technology in the �eld of health care andmedicine [32]. 3D printing, also known as additive manufacturing or additive rapid prototyping, canrapidly and accurately manufacture 3D stacked objects of complex shape under the control of thecomputer through the precise design of models [33]. At present, Stereolithography (SLA), FusedDeposition Modeling (FDM), Selective Laser Sintering (SLS), and Three-dimensional printing (3DP) arecommon methods in 3D printing [34]. Among them, SLS is a kind of technology that makes use ofpowder and computer-control to rapidly separate and produce objects. It uses a laser to combine powderparticles, building them layer by layer, and then gain them from under the powder bed [35-36]. Comparedwith FDM, SLS is a single-step process that does not require hot-melt extrusion to pre-produce suitable�laments and produces higher resolution ratio objects due to laser accuracy [37-39]. 3D printing isgradually rising in medicine. Brito [40] reported the advantages of additive manufacturing in mandibularfracture surgery. Mafeld [41] carried out the feasibility study of the 3D printing vascular model. Ren [42]used 3D printing to design a calcaneal surgical guide plates to improve the surgical treatment scheme.Tetsworth [43] applied 3D printing to the reconstruction of complex post-traumatic osteoplasty andachieved ideal results. Personalized biomedical materials can be prepared by 3D printing to meet theneeds of different patients. It has broad prospects in terms of tissue scaffold, cell printing, prosthesis.The 3D printing in the research and development of biomaterials involves the following key technologies:�rstly, digital three-dimensional Computer-Aided Design (medical prototype object simulation)system(CAD), which transforms CAD model into "clone" products of real tissues or organs; secondly,medical biomaterials with 3D printing; thirdly, the technology and equipment suitable for biomedicalmaterials printing. These are indispensable.

Although 3D printing has gained the attention of many domestic and foreign scholars, especially in thepreparation of biomedical materials, the clinical application of 3D printing still faces many challenges,which are still in the initial stage. The �rst problem is CAD software and the selection of 3D printing rawmaterials including the mechanical properties, biocompatibility and activity retention, etc. Secondly, if 3Dprinting is applied to living cells or tissues, the survival rate of cells on the surface or inside of the product

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needs to be maintained during the whole processing. Finally, we need to clarify the mechanism of celladhesion, growth, and differentiation inside the material.

To develop new-type material implants with independent intellectual property rights, reduce or eliminatethe dependence on imports, and provide Chinese with suitable prices, multiple choices, and betterperformance implants, Ta-Nb alloy implant taking Ta as the main component and adding the appropriateamount of Nb was prepared by domestic 3D printing equipment and SLS technology in the early stage. Inthis study, a traditional Ti implant was used as a control. The main research contents include the TensileStrength(TS) and Hardness analysis in vitro of Ta-Nb alloy implant. Effects of Ta-Nb alloy on the growth,proliferation, migration, early adhesion, and cell cycle of human Oral Mucosa Fibroblasts(hOMF). Thecontent of metal ions in the different extracting liquid was analyzed to determine the stability of metalmaterials. The research of this project is of great theoretical and practical signi�cance to improve China'sindependent innovation ability, promote the development of the 3D printing industry chain andprosthodontics.

ResultsGeneral observation of experimental materials

 Tantalum-Niobium alloy (TaNb40) implant material was prepared by SLS. SLS laser parameters were250W. Its size is 4.5mm in diameter, height 12.2mm, and weight 2.2g (without abutment). The shape issimilar to that of Ti implant commonly used in the clinic (Fig. 1 and Fig. 2). It can be seen from Fig. 1 thatthe surface of the TaNb40 alloy is bright gray with a certain degree of roughness, which is the originalshape that has not been surface treated. To facilitate the co-culture of experimental materials and cells,and the TaNb40 alloy disc prepared by SLS was used to replace the implant for an experiment in vitro.The surface of the substitute was bright silver (Fig. 3). There is little difference in appearance between Ta,Ti, and Ni metal samples prepared by the traditional casting method (Fig. 4).

The mechanical properties of TaNb40 implant

TaNb40 implant was tested by a Third-Party Inspection Agency and placed on a tensile tester to detectthe tensile property of the material, including TS (Mpa), Yield Strength (YS) (Mpa), and Elongation (%), aroutine test item of metal material. The results were shown in Table 1. The TS, YS, and Elongation ofTaNb40 implant were 548 ± 50MPa, 420 ± 30MPa, and 40%, respectively, besides, the Vickers hardnesswas 425HV.

Growth of Cells

The morphology of primary hOMF cultured to the 5th-6th generation is shown in Fig. 5, observed cellmorphology after recovery was brightly circled under an inverted microscope, and cells had well-de�nednuclear membranes. After 24 hours [Fig .5(a1)-(a3)], most of the cells emerged long fusiform, a few cellshad more branches, extended outward, and it changes like starlike. On the 3rd day [Fig.5(b1)-(b3)], the

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cells grew rapidly, increased number, the processes connected. On the 5th-7th day [Fig. 5(c1)-(c3) andFig.5(d1)-(d3)], the cell volume increased, and the processes fused into a network, increased cell densityand collagen matrix secretion could be observed clearly after HE staining. In this experiment, the 5th-6th

generation cells as samples grew well, whose morphology was consistent with that of normal �broblasts.

Direct effects of materials on cells

As shown in Fig.6, compared with the normal control in Fig.5, the number and morphology of cells of theco-culture with TaNb40, Ta, Ti, and Ni on the 1st, 3rd and 5th day were as follows: (1) On the 1st

day[Fig.6(a1)-(d1) and Fig.6(a2)-(d2)], the cells in the experimental group (TaNb40, Ta) and the positivecontrol group (Ti) grew well, a very few non-adherent cells were observed. There was little difference incell morphology among the three groups under the light microscope. However, only about 50% of the cellsadhered to the wall in the negative control group (Ni), the adherent cells were not in good shape, showingthe short rod-shaped structure and no polygonal cells. (2) On the 3rd day[Fig.6(a3)-(d3) and Fig.6(a4)-(d4)], the number of cells gradually increased, the processes were connected into reticular formation inthe TaNb40, Ta, and Ti group. On the 5th day[Fig.6(a5)-(d5) and Fig.6(a6)-(d6)], the cells in TaNb40 andTa groups were densely distributed around the materials, and a small amount of cell apoptotic fragmentswas observed. There was no cell attachment in the area about 0.1 mm around the Ti sheet, and the cellsoutside this region grew and arranged well. According to the cytotoxicity scoring standard in vitro, thescore of these three materials can be recorded as 0. In the Ni group, about 80% of the cells were roundwith no protruding pseudopodia, the number of cells decreased as the dissolution of some cells, and theabove morphological changes became more obvious over time, there were almost no normal adherentcells on the 5th day, the cytotoxicity was grade 4, which was material with severe cytotoxicity. (3) Thecells at different distances from the same material on the same day showed that there was no signi�cantdifference between TaNb40 and Ta at three-time points, but the number of cells far away from Ti seemedto be slightly more than that around the material. In the Ni group, it seems that the cells farther away fromthe material were in better condition at the same time than those near the material.

Effects of materials on cell early adhesion

The micrographs of adherent cells after AO �uorescence staining were shown in Fig.7. In the TaNb40 andTa group, the cell showed a large amount of clear green �uorescence, which was evenly distributed on thesurface of the material, and the number of adhesion cells increased signi�cantly with time. In the Tigroup, most showed red and yellow �uorescence, which represented the difference of binding capacitybetween staining agent and DNA. The cells were scattered in different visual �elds, and a small numberof cell fragments and apoptotic bodies could be seen, the amount at the 12th hour was signi�cantly morethan that at the 4th and 6th hour. In the Ni group, the quantity was signi�cantly less than that of the otherthree groups at each time point. It can be seen that the chromatin shrinks and breaks into patches ofvarying sizes, showing the shape of green fragments, and only a few cells adhered at three-time points.

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According to the quantitative analysis in Table 2 and Fig.8, the comparison of cell adhesion rate wassuccessively Ti > Ta > TaNb40 > Ni and the difference between Ti and Ta, Ti and TaNb40 was statisticallysigni�cant (P < 0.05). At the 6th and 12th hour, the cell adhesion rate successively was Ta > TaNb40 > Ti >Ni, there were signi�cant differences between Ta and Ti, TaNb40 and Ti (P < 0.01). The adhesion rateincreased with time in TaNb40, Ta and Ti group, and the highest at the 12th hour. No signi�cant differencebetween the 4th and 6th hour in the Ti group (P > 0.05), but it increased signi�cantly between the 6th and12th hour (P < 0.01). However, The adhesion rate of the Ni group was very low at all time points, and themean value of the TaNb40 and Ta group exceeded 100% at the 12th hour. It can be seen that bothTaNb40 and Ta have no effect on cell adhesion, and the initial adhesion process of surface cells waseven better than that of Ti, and can quickly adhere to the surface in the early stage.

Effects of materials on cell proliferation

To evaluate the effect of different material extracting liquid on cell proliferation, using CCK-8 reagent andenzyme labeling to detection. According to the quantitative analysis results in Fig.9, on the 1st day, thecell proliferation activity (OD value) was as follows: blank control > Ta > Ti > TaNb40 > Ni, Ta and Tigroup (P < 0.05), TaNb40 and Ti group (P < 0.01). On the 3rd day, the comparison was blank control > Ta >Ti > TaNb40 > Ni, there was statistical difference between Ti and Ta Group (P < 0.05), but there is notbetween Ti and TaNb40 group (P > 0.05). On the 5th day, the order successively was blank control group >Ti > Ta > TaNb40 > Ni, Ti and TaNb40 group (P < 0.01), Ti and Ta Group (P > 0.05). The OD value of theblank control group at each time point showed the highest cell activity (P < 0.01). There was no statisticaldifference in the OD value of the Ni group at three-time points. As can be seen from Fig.9, the number ofcells did not increase but also decreased with time.

The RGR and cytotoxicity grade as shown in Table 3 were calculated from the OD value. The Ta, TaNb40,and Ti group was grade 1, which met the requirements of medical biomaterials, while the Ni group wasgrade 3, which did not conform to the speci�cation of medical biomaterials.

Effects of materials on cell cycle

As shown in Fig.10, the cell cycle distribution was detected by �ow cytometry after 48 hours of treatmentwith different extracting liquid, and the data were listed in Table 4. The one-way ANOVA analysis on thepercentage of cells in G1, G2, and S phase among the �ve groups in Table 4 indicated there weresigni�cant differences among the phase of each cycle between groups(P < 0.01). However, comparedwith the blank control group, the percentage of G1 / S phase cells tended to increase and the G2 phasedecreased in the Ta Group. The TaNb40 group increased in the G1 phase and decreased in S / G2 phase,although the cell cycle percentage of the TaNb40 group was statistically different compared with a blankcontrol group, it tended to be similar in numerical value, and there was no signi�cant increase ordecrease. In the Ti group, the G1 phase decreased and S / G2 phase increased. In the Ni group, thepercentage of S-phase cells increased signi�cantly, and Ni induced S phase arrest accompanied by a

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decrease in the percentage of G1 phase cells. Compared with 3.11% of the blank control group, the cellstreated with Ni had the highest apoptosis percentage of 3.91% in the sub-G1 phase.

Detection of metal ion content

The content of metal ions in the four kinds of material extracting liquid was listed in Table 5. The amountof ions released by Ni was the highest, up to 264 mg/kg. The metal ions were hardly detected in othergroups. In the reference data of detection limit value, the amount of the ions of TaNb40 and Ta Groupwere less than 0.1mg/kg, and that of Ti group were less than 0.2mg/kg.

DiscussionAt present, Ta is widely used in the biomedical �eld, especially in orthopedic metal implants, whichattracts the attention of researchers in the �eld of biomaterials. In recent years, PTTM has beenincorporated into Ti alloy dental implants to improve osseointegration. Compared with traditional boneimplant materials, Ta has high corrosion resistance, hardness, and wear resistance, which can avoid theadverse biological effects to the most extent caused by the release of metal ions in vivo. On the otherhand, Ta has strong osteoinductive and osseointegration abilities in vivo, even better than Ti dentalimplants, which can improve the stability of implants at the early stage [44-46]. However, Ta is a relativelyactive element, the powder particle size is small, and it is easy to oxidize at high temperature, so it isdi�cult to prepare, at the same time, the mechanical strength and density of solid pure Ta are very high.Adding Nb element can improve the mechanical properties of the material, reduce the sinteringtemperature and manufacturing cost, and promote the sintering of Ta at high temperature, to meet theclinical requirements of dental implant material. Also, the traditional processing technology is di�cult toprepare Ta, therefore, SLS was used to meet the personalized requirements in the early stage of thisstudy. The surface of TaNb40 alloy prepared by SLS is brightly gray with a certain roughness, moreover,the oxygen content is lower than 40ppm in the degreasing environment, and there is no oxidationphenomenon. As a load-bearing part of the body, the bio-metal orthopedic implant must have goodmechanical properties. In addition to evaluating the stability of implant through bone integration, thematched mechanical properties between the alloy and bone tissue are conducive to the uniformdistribution of force transmission to the alloy and surrounding bone tissue, and prevent the generation of"stress shielding" [47]. Mechanical properties and biocompatibility are the primary factors to beconsidered in the design of new materials [48]. Especially in the dental implant, they will be exposed tothe alternating stress of load and fatigue when performing their functions [49]. The strength of the alloyshall be high enough to withstand external forces including tension, compression, bending and torsion[48, 50]. TS and YS are the basic properties of hard tissue replacement materials, which can prevent theplastic deformation of materials in the process of implantation, to ensure stability in bone tissue [51]. Thetechnical standard ASTM F67 divided pure Ti implant into four grades: G1-G4, with YS range of 170-483MPa and TS of 240-550 Mpa [51]. The YS of Ti alloy (Ti6Al4V) implant is signi�cantly higher than that ofpure Ti, and the YS of Ti alloy prepared by different processes is in the range of 360-3267 MPa [52]. TheYS and TS of TaNb40 alloy detected in this study were 420 ± 30 MPa and 548 ± 50 MPa respectively,

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compared with the pure Ti and Ti alloy recorded in the literature, these two aspects of TaNb40 implant arewithin the range of mechanical performance indicators, and have good mechanical properties. TheElongation of the TaNb40 alloy is 40%, which is higher than pure Ti and Ti alloy implant based on therelevant literature [51], indicating that the TaNb40 implant has the characteristic of not easy brittlefracture. Besides, compared with Ti material, the Vickers hardness value (425HV) of TaNb40 was higherthan that of pure Ti and Ti alloy implant [51]. Hardness refers to the resistance of the material topermanent distortion. It is not too much to say Hardness is an important indicator of wear resistance.Besides, the elastic modulus is also an important property, if it is far greater than that of bone tissue, thedifference may produce greater stress at the bone-implant interface in the process of load transmission,resulting in bone loss and implant loosening failure. In our previous studies, through a series ofoperations such as the design of computer-aided software, the preparation of SLS raw material powder,and the control of processing conditions, the TaNb40 implant was successfully prepared. In this study,the YS and Hardness were preliminarily determined. However, as a medical biomaterial implant, it is stillnecessary to further detect compressive stress, shear force, elastic modulus, and fatigue strength in thefuture because the stress in the jaw is very complex, which could provide the basis for adjusting theparameters of 3D printing properly so that all the mechanical properties of the implant can enough tomatch the bone tissue in vivo.

Good biocompatibility is the prerequisite and foundation of Ta application in the biomedical �eld. Atpresent, the evaluation of biocompatibility of biomaterials mainly refers to International Organization forStandardization (ISO) 10993 and national standard GB / T16886, including the utilization of differentexperimental methods in vitro and in vivo. Researchers can choose some of methods to achieve their ownresearch objectives. It can be concluded whether the material conforms to the biological safety andfunctionality through a series of evaluations, in other words, the material has no toxic effect on thehuman body and does not cause host heterologous recognition reaction. Besides, the biomaterials shouldbe able to perform the corresponding functions in the speci�c parts, not be rejected and destroyed,maintain their original physicochemical, mechanical, and biological properties, and have a long-termgood combination with the host. Therefore, the other focus of this study is the cytotoxicity of metalmaterials in vitro. The cytotoxicity test is one important index to evaluate the biocompatibility, which isthe most basic experimental method in biological performance evaluation of material. In this study, weobserved the effects of TaNb40 and Ta on the morphology, early adhesion, proliferation, and cell cycle ofhOMF in vitro, moreover, widely recognized metal pure Ti with good biocompatibility as positive controland pure Ni as negative control were used. After initial implant stability, it is also very necessary to closethe soft tissue wound in the second stage of implant surgery, which can promote the healing of softtissue and prevent the infection caused by a microorganism and other exogenous substances. Thehealing of oral mucosa soft tissue had to do with the �broblast proliferation and collagen deposition [53].Due to the poor antigenicity of �broblasts and the nonspeci�city of antigens, some studies tried toidentify �broblasts based on immunohistochemical staining, but the rate of success rate is very low[54-56]. Therefore, no immunohistochemical method was used to identify �broblasts in this study.Ultrastructurally, �broblasts are identi�ed by their stellate appearance with slender branching pseudopods

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and have marked rough endoplasmic reticulum(RER) and Golgi complex [57-58]. We observed the hOMFby light microscope, and the results were consistent with the morphology of normal �broblasts.

To direct observation of the number and morphological changes of the cells in contact with the materials,the direct contact method was used to detect the cytotoxicity. On the 5th day, a small number ofapoptotic fragments were observed around the TaNb40 Ta Ti group, which was the normal apoptosisduring the growth of cells. In the Ti group, there was no cell attachment in the area about 0.1 mm aroundthe metal sheet, because the weight of Ti (0.2756g) was less than that of Ta (0.6964g) and TaNb40(0.7007g) when inoculated with the same volume of culture medium with the same number of cells, Tiwould not adhere �rmly to the plate due to buoyancy, which affected the surrounding cells to a certainextent. According to the cytotoxicity scoring in vitro, TaNb40, Ta, and Ti were recorded as 0, which did notaffect cell growth and had no toxicity to cells. In the Ni group, about 80% of cells were suspended deadcells on the 5th day, the cytotoxicity score was 4, which was considered as a serious toxicity material.Due to the different number and morphology of cells in different distances from the same material, it canbe inferred that the cell is affected by some metal elements released from the material, which makes themetal ions distribute unevenly in the culture medium, thus affecting the growth of surrounding cells.Toxicity test is to detect the effects of small molecular substances on cells when materials are degradedor decomposed, the adverse reactions in local tissues are related to the metal ions released by material[59]. Therefore, the cytocompatibility in vitro can also be analyzed by the indirect contact, that is, theextracting liquid method. By ISO 10993-5:2009, the effects of TaNb40, Ta, Ti, and Ni extracting liquid oncell proliferation were tested. CCK-8 can react with enzymes in mitochondria of living cells and displayorange-yellow in solution, therefore, the more living cells, the greater the degree of reaction with reagent,the deeper the orange color. The higher the OD value indicating that the cytotoxicity of the material issmaller. The OD value measured by Microplate Reader can re�ect the cell activity and cell proliferation.The constant �uctuation of OD values of TaNb40, Ta, and Ti at three-time points may be theunintentional damage to cells caused by the gun head and the error of the testing instrument. Thestatistical difference between each of the three group and the blank control group indicates that eitherthese three materials do have a slight impact on cells, or the initial number of cells in the blank controlgroup is more than that in the TaNb40, Ta and Ti group due to the counting error, so go a step furtheranalysis the cell cycle distribution. Although there was the statistical difference, the cytotoxicity analysisshowed that TaNb40, Ta, and Ti did not produce cytotoxicity, and the grade was 1, which met therequirements of medical biomaterials. However, the OD value of the Ni group was lower than 0.5 at theoutset, and the cell proliferation was inhibited over time, which was consistent with the results of directcontact morphological analysis. The cytotoxicity of the Ni group was grade 3, which did not conform tothe standard of medical biomaterials.

The surface properties of the material, such as surface energy, hydrophobicity/hydrophilicity, net charge,and roughness, affect the adsorption of cells and macromolecules on the surface after implantation [60].To observe the effect of the surface properties on the early adhesion of hOMF, an AO reagent was used tostain cells and observe the early cell adhesion at the 4th, 6th, and 12th hour. AO is a �uorescent dye used

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to observe the number of adhesion cells on the surface of opaque material, the reagents emit differentcolors of �uorescence by binding with nuclear DNA and RNA, so the adhesion ability of cells can beevaluated. The cell adhesion rate of TaNb40, Ta, and Ti group increased with time, and the maximumwas found at the 12th hour, the TaNb40 and Ta increased signi�cantly at three-time points (P < 0.01),indicating that they did not affect the cell adhesion. Compared with the other three groups, the initialadhesion rate of the Ni group was very low, and as time goes on, not only no cells attached, but also thecells adhering on the surface of the material also showed lysis apoptosis, and the adhesion rate waslower. At the 4th hour, the cell adhesion rate of the Ti group was higher than that of TaNb40 and Ta group(P < 0.05), at the 6th and 12th hour, the TaNb40 and Ta were higher than that of the Ti group, and thedifference was statistically signi�cant (P < 0.01), which make clear that the initial adhesion of cells of theTaNb40 and Ta is better than that of Ti, and can quickly stick to the surface in the early stage, this resultmay be related to the roughness surface prepared by SLS technology. Studies have shown that thesurface topography (�at, rough, nanometer) and crystal size of biomaterials affect the interactionbetween cell and material interface, and then have an effect on their biological characteristics [61]. Thelow level of cell differentiation in the �at morphology might be due to its more inert surface, resulting inless cellular reaction [62]. The roughness of the nanoparticle size can intervene in the behavior of thecells. Some scholars [63-64] prepared bulk ultra�ne-grained pure Ta by Equal Channel Angular Pressure(ECAP), after treatment, the number of grain boundaries on the surface increased, providing more celladhesion sites, showing better cell activity and biocompatibility. The nano pits can change the surfaceroughness, wettability, and the adsorption capacity of �bronectin, it could also be recognized by�broblasts, especially when the size of nano pits is 50 nm and 60 nm respectively, which can signi�cantlyenhance the attachment and proliferation of �broblasts [65]. The TaNb40 surface showed a certainroughness whose pit size could not be determined due to without measurement of the surfacemicrostructure. It can be seen that the early adhesion rate of cells on the rough surface of TaNb40prepared by SLS is higher than that on the Ti surface from our experimental results, but the error in theoperation is also not ruled out, which is related to the inherent chemical properties of Ta surface. Somescholars have studied the effect of inherent chemical properties of Ti and Ta on Bone MarrowMesenchymal Stem Cells (BMSCs), the results showed that the expression of integrin α5 and β1 on Tasurface was higher. Integrin plays an important role in the formation of focal adhesion complex, mediatesintracellular signal transduction, and regulates the process of cell differentiation [66]. Other studies haveshown that [44-46] Ta has better osteogenic differentiation than Ti in prosthesis and implant coating. Thesurface characteristics of materials, such as surface structure and physicochemical properties, cansigni�cantly affect the behavior of cells and subsequently bone induction and osseointegration. At the12th hour, the average number of adherent cells on the Ta and TaNb40 group was more than 100%,indicating that not only the number of adherent cells on the surface of the material increased signi�cantlybut also some cells were in the early stage of proliferation at this time point. In a word, the TaNb40implant or disk-shaped material made by SLS has no surface treatment, which does has better celladhesion, even better than Ti. Therefore, it can be inferred that it may not require further surfacetreatment, and the relatively rough surface is conducive to cell adhesion and growth.

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To determine whether the cell cycle was blocked by different materials and which cell cycle stage isinhibited by Ni, the cells were stained with Propidium Iodide (PI), so the DNA content was detected by �owcytometry. Various cell cycle stages have different DNA ploidy numbers, according to the distribution ofcells in each phase of the cell cycle, we can judge whether the materials have no, promote or inhibitproliferation effect on cells. The cells were treated with the extracting liquid after 48 hours. Comparedwith the blank control group, the percentage of G1 / S phase cells in the Ta group tended to increase, theG2 phase decreased, indicating that DNA synthesis accelerated, thus promoting DNA replication and cellproliferation. In the TaNb40 group, the results indicated that the cells were in normal growth andproliferation stage. In the Ti group, the G1 phase decreased and S / G2 phase increased, the cells were inG1 to S phase transformation stage combined with cell proliferation results, which promoted cellproliferation. The cell proliferation results between the TaNb40, Ta, Ti, and blank control at three- timepoints were statistically different (P < 0.01), but there was no cell cycle arrest. The possible reason is thatthe initial number of cells in the blank control group was more than that in these three groups due to theerror of cell count, thereby displaying the statistical difference of OD value in each group. Compared with3.11% of the blank control group, the percentage of S-phase cells increased signi�cantly in the Ni group,and 3.91% of cells in the sub-G1 phase were apoptotic. Therefore, Ni induced cell cycle arrest in the Sphase and promoted cell apoptosis in this study. The ion content of Ni extracting liquid was 264mg/kg.Qiao et al. [67] found that 10 um Ni could cause DNA damage in vitro. Besides, it also was found that [68]Ni nanoparticles (NiNPs, size: 28nm) decreased the survival rate of human liver (HepG2) cells in a dose-dependent manner in the concentration range of 25-100μg / ml. DNA damaged cells accumulate at Gap1(G1), DNA synthesis (s), or Gap2 / Mitosis (G2/M). Studies have shown that [68-69] Nickel chloride(NiCl2) induces G2 / M arrest of the liver. A large number of metal ions accumulated in the body caninduce a series of pro-in�ammatory reactions. A kind of metal ion can activate a variety of intracellularsignaling pathways to mediate the release of cytokines, promote the formation of the local in�ammatoryresponse and initiate cell apoptosis mediated by mitochondria. If this immune- in�ammatory reactionpersists, osteolysis will eventually occur, resulting in implant loosening and fracture. The long-term wearof arti�cial joints and the particles produced by dissociation in the humoral environment will lead to theabove phenomenon. In this study, the ion contents in the different extracting liquids were furthermeasured. Except for Ni ions, almost no metal ions were detected in other groups. The amount of metalions precipitated in TaNb40 and Ta group was less than 0.1mg/kg, and the Ti was less than 0.2mg/kg.The number of the released metal elements is closely related to the properties of surface passivationoxide �lm and corrosion resistance. Many references [70-72] have proved that the passivation oxide �lmon the surface of Ta is very stable, and no corrosion sign has been found in physiological solution invitro. Two kinds of passive oxide �lms, Ta2O5 and Nb2O5, exist on the surface of TaNb40, which hasexcellent corrosion resistance [72]. The corrosion resistance of Ni is very poor, the stability of surface �lmNiO is low, and a large amount of Ni ion [73-74] can be released in a short time. Therefore, Ta and TaNb40have excellent corrosion resistance and biocompatibility. At least there is almost no metal ion release invitro. The poor biocompatibility of Ni is related to the instability of surface passive �lm and the release ofNi.

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ConclusionsTaNb40 implant was prepared by SLS technology of 3D printing in our previous studies, its surface isbright gray, showing a certain degree of roughness. (1) The TS, YS, Elongation, and Vickers hardness was548 ± 50MPa, 420 ± 30MPa, 40%, and 425HV respectively, which indicate that the TaNb40 implant hasgood mechanical properties, wear-resistance, and not easy to brittle fracture. (2) TaNb40 and Ta weretested in vitro by direct contact, AO �uorescence staining, CCK-8, and �ow cytometry, it was found that thetwo materials did not inhibit cell proliferation and cell cycle arrest, the cytotoxicity grade in vitro was 1.There was almost no metal ion release in the culture medium, and both of them have good early celladhesion ability. All above these, it meets the requirements of medical biomaterials. (3) In general, themechanical properties and cell compatibility in vitro of TaNb40 are excellent, which is expected to replacethe Ti implant prepared by the traditional casting method. It is of guiding signi�cance for the researchand development of implant materials with independent intellectual property rights.

However, the stress on the implant in the jaw is very complex, which is still necessary to detect thecompressive stress, shear force, elastic modulus, fatigue strength in the future, to provide the basis fortimely adjusting the parameters of 3D printing, and make the mechanical properties of the implant matchthe bone tissue in vivo. In terms of the biological performance evaluation, the osteogenic of cells on thesurface of materials and the osseointegration in animals also need to be further studies in the future.

Preparation of experimental materials

The CT Scanning (FS271M, Hunan Huashu High Tech Co., Ltd) appearance parameters of the TaNb40sample (Changsha Nanfang Tantalum Niobium CO, LTD, China) was set according to commonly used Tialloy (Basic implant system, USA) implant in the clinic. The chemical composition of TaNb40 suitable forSLS in this experiment is Ta: Nb = 5.8 ~ 6.2 : 3.8 ~ 4.2. The composition and proportion of residualelements are shown in Table 6, and the totality weight ratio is less than 1.5%, the particle size of Ta andNb powder is 10μm-60μm, and the SLS laser power is 250W. To facilitate the co-culture of the cells withmaterials in the same culture dish, the Ta-Nb alloy sheet, or TaNb40 for short (9 mm in diameter and 1mm in thickness) was prepared by SLS. At the same time, Ta sheet with the same size was prepared bytraditional casting method as another experimental group, and Ti and Ni sheet (Yudingda Metal, China)was used as positive and negative control materials respectively. The materials were sterilized by hightemperature and high pressure.

Characterization of materials

In this project, the test of mechanical properties of TaNb40 includes TS and Vickers hardness. Fivesamples were used for each index. According to the national standard GB / T1040, the tensile propertiesof TaNb40 alloy were tested by a Tensile testing machine (Shanghai Hesheng Instrument Technology Co.,Ltd) at room temperature. Besides, the unit stress on the indentation area was measured by Vickershardness tester (Shanghai Jujing Precision Instrument Manufacturing Co., Ltd) according to the GB / T4340.1-2009.

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Primary culture of human Oral Mucosa Fibroblasts

This study was approved by the ethics committee of Xiangya School of Stomatology, Central SouthUniversity, with the approval number of 20190015. The human Oral Mucosa Fibroblasts (hOMF) strain(set up by the author's team) were established as experimental cells. The third generation of hOMF wastaken out from liquid nitrogen and the cells after resuscitation were cultured in an incubator(ThermoFisher) containing 5% CO2 and 37 ℃ / 95% humidity until it grows to 80% - 90%, then goingdown to the future generation.

The cell growth observed by the direct contact method

The experiment was divided into �ve groups: TaNb40, Ta, Ti, Ni, and blank control group (only cellswithout material). It was proper in the density of 3×104 cell/ml, each group of materials setting up fourparallel groups was placed in a 24-pore plate (Excell Bio, China) to co-culture with the cells. On the 1st, 3rd,and 5th day, the cell growth around the material was observed by an inverted microscope(Leica).According to the national standard GB / T 16886.5, the cytotoxicity in vitro score was recorded taking100% of the number of cells in the blank control group as the benchmark. Such as the following: 0. Nocytotoxicity, no effect on cell growth; 1. Slight cytotoxicity, the number of round cells ≤ 20%; 2. 20% < thenumber of round cells ≤ 50%, with slight cell growth inhibition; 3. 50% < the number of rounds ordissolved cells ≤ 70%; 4. Severe cytotoxicity, resulting in serious cell damage.

The early cell adhesion observed by AO �uorescence staining

The experiment was divided into four groups: TaNb40, Ta, Ti, and Ni group. Acridine Orange (AO)�uorescence staining (BestBio, China) was used to observe the early cell adhesion on the surface of thematerial (within 12 hours). Collecting the sample cells, counting, the cell density was about 2 × 105/ml,and the 50ul cell suspension was added to the different material surfaces. After 30 minutes of cellsedimentation, adding 0.5ml culture medium again. The 50 ul of dye solution was added to the surfaceafter the 1st day and 4th day respectively and incubated for 10-15 minutes in dark at 4 ℃. The maximumexcitation wavelength was 488 nm.

Using Image J software, �ve visual �elds were randomly selected for each sample under the microscope,�nally set up the database, perform statistical analysis.

Prepare the extracting liquid

According to the national standard ISO 10993-5:2009, the material extracting liquid was preparedaccording to the ratio of the extraction medium and the surface area (or weight) of the biomaterial. In thisexperiment, the sample diameter is 9mm and the thickness is 1mm, the extraction ratio is 3 / cm2 · ml-1

based on Table 7. It is calculated that 0.53ml culture medium should be added to the 24-well platecontaining materials, and placed in the refrigerator(ThermoFisher) at 4 ℃ as the extracting liquid.

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The cell proliferation determined by CCK-8

The experiment was divided into �ve groups: the extracting liquid of TaNb40, Ta, Ti, Ni, and the blankcontrol group. The cell density was about 3×104 cells/ml. Each group of materials was put into a 96-wellplate (Excell Bio, China), dropping 100ul cell suspension, after 24 hours, discarding the old culturemedium, and adding 100μl per experiment group. After the 1st, 3rd, and 5th day, add 10ul CCK-8 reagent(BestBio, China).

The OD value of each pore was measured by enzyme-labeled instrument (Thermo Scienti�c MultiskanFC) at 450 nm. The OD value of the blank control group was taken as the standard, and the relativegrowth rate (RGR) was calculated by the ratio of OD mean value of experiment groups to that of the blankcontrol group. RGR (%) = ODsample / ODblank × 100%. The cytotoxicity evaluation grading is shown inTable 8.

Cell cycle determined by �ow cytometry

The experimental groups were the extracting liquid of TaNb40 and Ta, the control groups were theextracting liquid of Ti, Ni, and the blank control group(the complete medium). The number of cells in eachgroup was about (5-10) × 106. The 3.5 ml of cold ethanol (Excell Bio, China) was slowly dropped intoeach tube(Excell Bio, China) with cells, and the �nal concentration of ethanol was about 75%. Thesamples were �xed overnight at 4 ℃ and stored at - 20 ℃. Within one week, the 400 UL PI stainingsolution(BestBio, China) was added to resuspend the cells. Results were detected by �ow cytometry(BD,USA), the excitation wavelength was 488 nm, and Cell DNA content, light scattering were analyzed byModFit software.

Determination of metal ions in the extracting liquid

 The contents of metal ions in each group were determined by Inductively Coupled Plasma massspectrometry (ICP-MS) (NCS Testing Technology Co., Ltd). The text process was shown in Fig. 11.

AbbreviationsSS: Stainless Steel; Ti: Titanium; TiZr: Titanium-Zirconium; Al: Aluminum; V: Vanadium; Ta: Tantalum;PTTM: Porous Tantalum Trabecular Metal; Nb: Niobium; SLA: Stereolithography; FDM: Fused DepositionModeling; SLS: Selective Laser Sintering; 3DP: Three-dimensional printing; CAD: Computer-Aided Design;TS: Tensile Strength; hOMF: human Oral Mucosa Fibroblasts; AO: Acridine Orange; RGR: Relative GrowthRate; ICP-MS: Inductively Coupled Plasma Mass Spectrometry; YS: Yield Strength; ISO: InternationalOrganization for Standardization; RER: Rough Endoplasmic Reticulum; ECAP: Equal Channel AngularPressure; BMSCs: Bone Marrow Mesenchymal Stem Cells; PI: Propidium Iodide.

Declarations

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Acknowledgments

Thanks to Xiangya Medical Science Research Center of Central South University for the technical supportof this study.

Funding

This research was funded by “2017 Key Research and Development Program of Hunan Province inChina(Grant. No.2017GK2265)”, “2018 Key Research and Development Program of Hunan Province inChina(Grant. No.2018SK2104)” and “Department of health project of Hunan Province in China, grantnumber (Grant. No.B2015-142).

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding authoron reasonable request.

Author Contributions

methodology, writing—original draft preparation, writing—review and editing, data curation, Huiling Li;

Conceptualization, validation, Junhui Huang and Zhigang Yao;

resources, visualization, Jian Zhang;

All authors have read and agreed to the published version of the manuscript.

Ethics approval and consent to participate

This study was approved by the ethics committee of Xiangya School of Stomatology, Central SouthUniversity, with the number 20190015.

Consent for publication

Not applicable.

Competing interests

The author(s) declared no potential con�icts of interest concerning the research, authorship, and/orpublication of this article.

Patents

Part of the work in this study has applied patent for invention: (1) A 3D-printing method of Tantalum-Niobium alloy dental implant, patent number: 201910208621.1; (2) A Tantalum-Niobium alloy dental

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implant and preparation, patent number: 201910208701.7; (3) A Tantalum-Niobium alloy dental implantmaterial and a Tantalum-Niobium alloy dental implant, patent number: 201910209344.6.

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Figures

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Figure 1

Ta-Nb alloy mplant prepared by SLS

Figure 2

Titanium alloy impiant commonly used in the clinic

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Figure 3

The TaNb40 alloy disc made by SLS

Figure 4

The Ta, Ti, and Ni experimental materials used by radibonal casting method

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Figure 5

The hOMF of the 5” - 6” generation, (a1)-(d1) (x 100) show the cell morphology under an invertedmicroscope ; (a2)-(d2) (x40) and (a3)-(d3) (x 100) show the cell morphology under a bght microscope

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Figure 6

The cells growth around different materials on the 1st, 3rd, and 5th day, (a1)-(d1) (a3)-(d3) (a5)-(d5) showcells around materials; (a2)-(d2) (a4)-(d4) (a6)-(d6) show cells relatively far away fom materials

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Figure 7

The AO �uorescence micrograph of cells on the different material surface at 4h, 6h, and 12h

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Figure 8

Comparison of the cells early adhesion rate on the different material surtace at 4h, 6h. and 12h (n=5, x ±S, *P<0.05. **P<0.01)

Figure 9

The Comparison of OD value in different extracting liquid for the 1st, 3rd, and 5th day (n=6, x ± S,*P<0.05. **P<0.01)

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Figure 10

The cells cycle distribution in different extracting liquid after 48h, (a)Ta; (b) TaNo40: (c) Ti; (d) Ni (e) blankcontrol

Figure 11

The ICP-MS Gatection process